Atherosclerosis is the major causative factor of heart disease and stroke, and the leading cause of death in Western countries is cardiovascular disease. Dyslipidaemia is a primary contributor to atherosclerosis. Because triglycerides are insoluble in the bloodstream, they are packaged for plasma transport into micelle-like lipoprotein particles composed of protein and phospholipid shells surrounding a non-polar core of acylglycerols, free cholesterol, and cholesterol esters. Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons (which transport dietary lipids from intestine to tissues); very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low density lipoproteins (LDL), (all of which transport triacylglycerols and cholesterol from the liver to tissues); and high density lipoproteins (HDL) (which transport endogenous cholesterol from tissues to the liver, as well as mediating selective cholesteryl ester delivery to steroidogenic tissues). All of these particles undergo continuous metabolic processing and have somewhat variable properties and compositions. Plasma concentrations of LDL and HDL are directly and inversely related, respectively, to the risk of atherosclerotic cardiovascular disease (Krieger, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 4077-4080).
HDL protect the arterial wall from the development of atherosclerosis by promoting efflux of excess cholesterol from cells in the arterial wall and returning it to the liver for excretion into the bile, as well as by protecting LDL from oxidation, thereby reducing the inflammatory response of epithelial cells, inhibiting the coagulation pathway, and promoting the availability of nitric oxide. The metabolism of HDL is influenced by several members of the triacylglycerol (TG) lipase family of proteins, which hydrolyze triglycerides, phospholipids and cholesteryl esters, generating fatty acids to facilitate intestinal absorption, energy production or storage. Of the TG lipases, lipoprotein lipase (LPL) influences the metabolism of HDL cholesterol by hydrolyzing triglycerides in triglyceride-rich lipoproteins, resulting in the transfer of lipids and apolipoproteins to HDL and is responsible for hydrolyzing chylomicron and VLDL in muscle and adipose tissues. Hepatic lipase (HL) hydrolyzes HDL triglyceride and phospholipids, generating smaller, lipid-depleted HDL particles, and plays a role in the uptake of HDL cholesterol (Jin et al., Trends Endocrinol. Metab., 2002, 13, 174-178; Wong and Schotz, J. Lipid Res., 2002, 43, 993-999). Endothelial lipase (also known as EDL, EL, LIPG, endothelial-derived lipase and endothelial cell-derived lipase) was identified using differential display to isolate mRNAs which were differentially regulated in response to oxidized-LDL (Jaye et al., Nat. Genet., 1999, 21, 424-428). Independently, the human endothelial lipase gene was identified in human umbilical vein endothelial cells (HUVECs) undergoing tube formation in a model of vascular formation (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175).
In humans, the endothelial lipase gene has been assigned to chromosome 18, and the rat endothelial lipase gene was identified and mapped to rat chromosome 18 in the vicinity of a quantitative trait locus that affects serum HDL levels after a high fat diet (Bonne et al., DNA Seq., 2001, 12, 285-287).
At least 50% of the variation in HDL cholesterol levels is genetically determined. The phenotype of elevated HDL cholesterol is often dominantly inherited, but homozygous deficiency of HL or of the cholesteryl ester transfer protein (CETP), which result in elevated HDL cholesterol, are recessive conditions. Recently, several genetic variations in the human endothelial lipase gene have been identified, 6 of which potentially produce functional variants of the protein, and the frequencies of these variants were found to be associated with elevated levels of HDL cholesterol in human subjects (deLemos et al., Circulation, 2002, 106, 1321-1326).
Notably, the endothelial lipase-mediated binding and uptake of HDL particles and the selective uptake of HDL-derived cholesterol esters have been reported to be independent of its enzymatic lipolytic activity (Strauss et al., Biochem. J., 2002).
Recombinant endothelial lipase protein has substantial phospholipase activity but has been reported to have less hydrolytic activity toward triglyceride lipids (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175; Jaye et al., Nat. Genet., 1999, 21, 424-428). However, endothelial lipase does exhibit triglyceride lipase activity ex vivo in addition to its HDL phospholipase activity, and endothelial lipase was found to hydrolyze HDL more efficiently than other lipoproteins (McCoy et al., J. Lipid Res., 2002, 43, 921-929). Overexpression of the human endothelial lipase gene in the livers of mice markedly reduces plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I (apoA-I) (Jaye et al., Nat. Genet., 1999, 21, 424-428).
On the basis of its amino acid sequence homology to other members of the TG lipase family, including the presence of a characteristic 19-amino acid “lid” domain predicted to form an amphipathic helix covering the catalytic pocket of the enzyme and confers substrate specificity to the enzymes of the TG lipase family, and its demonstrated phospholipase activity, the endothelial lipase protein is believed to be involved in lipoprotein metabolism and vascular biology (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175; Jaye et al., Nat. Genet., 1999, 21, 424-428).
Endothelial lipase was cloned from epithelial cells but has been demonstrated to be abundantly expressed in a variety of tissues including ovary, testis, thyroid gland, liver, lung, kidney and placenta, the latter suggesting the potential for a role in development (Hirata et al., J. Biol. Chem., 1999, 274, 14170-14175; Jaye et al., Nat. Genet., 1999, 21, 424-428). Interestingly, endothelial lipase mRNA levels were upregulated in HUVEC and coronary artery endothelial cells upon treatment with inflammatory cytokines implicated in vascular disease etiology and vascular remodeling, including TNF-alpha and IL-1beta. Thus, endothelial lipase is predicted to be intricately involved in modulating vessel wall lipid metabolism and to play a role in vascular diseases such as atherosclerosis (Hirata et al., Biochem. Biophys. Res. Commun., 2000, 272, 90-93).
Disclosed and claimed in U.S. Pat. No. 6,395,530 is an isolated nucleic acid which hybridizes at high stringency to a nucleic acid having a sequence selected from a group of which endothelial lipase is a member or to a target consisting of nucleotides from 44-79 of the endothelial lipase gene wherein the complement of said isolated nucleic acid encodes a polypeptide having triacylglycerol lipase or phospholipase A activity. Further claimed are a vector, a composition, a recombinant cell and method for preparing a polypeptide. Antisense nucleic acids are generally disclosed (Jaye et al., 2002).
Disclosed and claimed in PCT Publications WO 01/40466 and WO 00/73452 is an isolated nucleic acid having at least 80% nucleic acid sequence identity to a nucleotide sequence that encodes the endothelial lipase protein or to a nucleotide sequence selected from a group of which the endothelial lipase gene is a member, a vector, a host cell, a process for producing a polypeptide, an isolated polypeptide, a chimeric molecule, and antibody, a method of detecting said polypeptide in a sample, a method of linking a bioactive molecule to a cell expressing said polypeptide, a method of modulating at least one biological activity of a cell expressing said polypeptide, methods for stimulating the release of TNF-alpha from human blood, for modulating the uptake of glucose or FFA by skeletal muscle or adipocyte cells, for stimulating the proliferation or differentiation of chondrocyte cells, for stimulating the proliferation of inner ear utricular supporting cells, endothelial cells or T-lymphocyte cells, for stimulating the proliferation of or gene expression in pericyte cells, for stimulating the release of proteoglycans from cartilage, for stimulating the release of a cytokine from PBMC cells, for inhibiting the binding of A-peptide to factor VIIA, for inhibiting the differentiation of adipocyte cells, for detecting the presence of tumor in an mammal, an oligonucleotide probe derived from any of several nucleotide sequences cited, a composition useful for the treatment of immune related diseases, use of a polypeptide to prepare said composition, a method of diagnosing an immune related disease in a mammal, an immune related disease diagnostic kit, a method for identifying an agonist or a compound capable of inhibiting the expression and/or activity of a polypeptide, a vector, and an ex vivo producer cell. Antisense oligonucleotide agonists or antagonists are generally disclosed (Ashkenazi et al., 2000; Baker et al., 2001).
Disclosed and claimed in PCT Publication WO 01/96388 is an isolated polynucleotide comprising a sequence selected from a group of nucleotide sequences, complements of said sequences, sequences consisting of at least 20 contiguous residues of one of said sequences, sequences that hybridize to said sequences, sequences having at least 75% identity to said sequence, and degenerate variants of said sequence, an isolated polypeptide, an expression vector, a host cell, an isolated antibody, a method for detecting the presence of a cancer in a patient, a fusion protein, an oligonucleotide that hybridizes to said sequence, a method for stimulating and/or expanding T cells specific for a tumor protein, an isolated T cell population, a composition comprising a first component selected from the group consisting of physiologically acceptable carriers and immunostimulants, and a second component selected from the group consisting of said polypeptides, polynucleotides, antibodies, fusion proteins, T cell populations and antigen presenting cells that express a polypeptide, a method for stimulating an immune response in a patient, a method for the treatment of a cancer in a patient, a method for determining the presence of a cancer in a patient, a diagnostic kit comprising at least one oligonucleotide or antibody, and a method for inhibiting the development of a cancer in a patient. Antisense oligonucleotides are generally disclosed (Jiang et al., 2001).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of endothelial lipase.
Consequently, there remains a long felt need for agents capable of effectively inhibiting endothelial lipase function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of endothelial lipase expression.
The present invention provides compositions and methods for modulating endothelial lipase expression.